Successive passaging of a plant-associated microbiome reveals robust habitat and host genotype-dependent selection

AbstractThere is increasing interest in the plant microbiome as it relates to both plant health and agricultural sustainability. One key unanswered question is whether we can select for a plant microbiome that is robust after colonization of target hosts. We used a successive passaging experiment to address this question by selecting upon the tomato phyllosphere microbiome. Beginning with a diverse microbial community generated from field-grown tomato plants, we inoculated replicate plants across five plant genotypes for four eight-week long passages, sequencing the microbial community at each passage. We observed consistent shifts in both the bacterial (16S amplicon sequencing) and fungal (ITS amplicon sequencing) communities across replicate lines over time, as well as a general loss of diversity over the course of the experiment suggesting that much of the naturally observed microbial community in the phyllosphere is likely transient or poorly adapted. We found that both host genotype and environment shape microbial composition, but the relative importance of genotype declines through time. Furthermore, using a community coalescence experiment, we found that the bacterial community from the end of the experiment was robust to invasion by the starting bacterial community. These results highlight that selecting for a stable microbiome that is well adapted to a particular host environment is indeed possible, emphasizing the great potential of this approach in agriculture and beyond.Significance StatementThere is great interest in selecting for host-associated microbiomes that confer particular functions to their host, and yet it remains unknown whether selection for a robust and stable microbiome is possible. Here, we use a microbiome passaging approach to measure the impact of host-mediated selection on the tomato phyllosphere (above ground) microbiome. We find robust community selection across replicate lines that is shaped by plant host genotype in early passages, but changes in a genotype-independent manner in later passages. Work such as ours is crucial to understanding the general principles governing microbiome assembly and adaptation, and is widely applicable to both sustainable agriculture and microbiome-related medicine.

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Successive passaging of a plant-associated microbiome reveals robust habitat and host genotype-dependent selection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1908600116 ◽

2019 ◽

Vol 117 (2) ◽

pp. 1148-1159 ◽

Cited By ~ 19

Author(s):

Norma M. Morella ◽

Francis Cheng-Hsuan Weng ◽

Pierre M. Joubert ◽

C. Jessica E. Metcalf ◽

Steven Lindow ◽

...

Keyword(s):

Microbial Community ◽

Bacterial Community ◽

Amplicon Sequencing ◽

Host Genotype ◽

Microbial Composition ◽

Internal Transcribed Spacer Region ◽

Tomato Plants ◽

16S Amplicon Sequencing ◽

Host Evolution ◽

Plant Microbiome

There is increasing interest in the plant microbiome as it relates to both plant health and agricultural sustainability. One key unanswered question is whether we can select for a plant microbiome that is robust after colonization of target hosts. We used a successive passaging experiment to address this question by selecting upon the tomato phyllosphere microbiome. Beginning with a diverse microbial community generated from field-grown tomato plants, we inoculated replicate plants across 5 plant genotypes for 4 45-d passages, sequencing the microbial community at each passage. We observed consistent shifts in both the bacterial (16S amplicon sequencing) and fungal (internal transcribed spacer region amplicon sequencing) communities across replicate lines over time, as well as a general loss of diversity over the course of the experiment, suggesting that much of the naturally observed microbial community in the phyllosphere is likely transient or poorly adapted within the experimental setting. We found that both host genotype and environment shape microbial composition, but the relative importance of genotype declines through time. Furthermore, using a community coalescence experiment, we found that the bacterial community from the end of the experiment was robust to invasion by the starting bacterial community. These results highlight that selecting for a stable microbiome that is well adapted to a particular host environment is indeed possible, emphasizing the great potential of this approach in agriculture and beyond. In light of the consistent response of the microbiome to selection in the absence of reciprocal host evolution (coevolution) described here, future studies should address how such adaptation influences host health.

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Applications of the indole-alkaloid gramine modulate the assembly of individual members of the barley rhizosphere microbiota

PeerJ ◽

10.7717/peerj.12498 ◽

2021 ◽

Vol 9 ◽

pp. e12498

Author(s):

Mauro Maver ◽

Carmen Escudero-Martinez ◽

James Abbott ◽

Jenny Morris ◽

Pete E. Hedley ◽

...

Keyword(s):

Indole Alkaloid ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Dependent Manner ◽

Host Genotype ◽

Independent Manner ◽

Molecular Determinants ◽

Breeding Selection ◽

The Impact ◽

Soil Interface

Microbial communities proliferating at the root-soil interface, collectively referred to as the rhizosphere microbiota, represent an untapped beneficial resource for plant growth, development and health. Integral to a rational manipulation of the microbiota for sustainable agriculture is the identification of the molecular determinants of these communities. In plants, biosynthesis of allelochemicals is centre stage in defining inter-organismal relationships in the environment. Intriguingly, this process has been moulded by domestication and breeding selection. The indole-alkaloid gramine, whose occurrence in barley (Hordeum vulgare L.) is widespread among wild genotypes but has been counter selected in several modern varieties, is a paradigmatic example of this phenomenon. This prompted us to investigate how exogenous applications of gramine impacted on the rhizosphere microbiota of two, gramine-free, elite barley varieties grown in a reference agricultural soil. High throughput 16S rRNA gene amplicon sequencing revealed that applications of gramine interfere with the proliferation of a subset of soil microbes with a relatively broad phylogenetic assignment. Strikingly, growth of these bacteria appeared to be rescued by barley plants in a genotype- and dosage-independent manner. In parallel, we discovered that host recruitment cues can interfere with the impact of gramine application in a host genotype-dependent manner. Interestingly, this latter effect displayed a bias for members of the phyla Proteobacteria. These initial observations indicate that gramine can act as a determinant of the prokaryotic communities inhabiting the root-soil interface.

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SARS-CoV-2 does not have a strong effect on the nasopharyngeal microbial composition

Scientific Reports ◽

10.1038/s41598-021-88536-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tzipi Braun ◽

Shiraz Halevi ◽

Rotem Hadar ◽

Gilate Efroni ◽

Efrat Glick Saar ◽

...

Keyword(s):

Clinical Microbiology ◽

Amplicon Sequencing ◽

Viral Disease ◽

Nasopharyngeal Swab ◽

Microbial Composition ◽

16S Amplicon Sequencing ◽

The World ◽

Health And Disease ◽

Longitudinal Sampling ◽

Potential Interactions

AbstractThe coronavirus disease 2019 (COVID-19) has rapidly spread around the world, impacting the lives of many individuals. Growing evidence suggests that the nasopharyngeal and respiratory tract microbiome are influenced by various health and disease conditions, including the presence and the severity of different viral disease. To evaluate the potential interactions between Severe Acute Respiratory Syndrome Corona 2 (SARS-CoV-2) and the nasopharyngeal microbiome. Microbial composition of nasopharyngeal swab samples submitted to the clinical microbiology lab for suspected SARS-CoV-2 infections was assessed using 16S amplicon sequencing. The study included a total of 55 nasopharyngeal samples from 33 subjects, with longitudinal sampling available for 12 out of the 33 subjects. 21 of the 33 subjects had at least one positive COVID-19 PCR results as determined by the clinical microbiology lab. Inter-personal variation was the strongest factor explaining > 75% of the microbial variation, irrespective of the SARS-CoV-2 status. No significant effect of SARS-CoV-2 on the nasopharyngeal microbial community was observed using multiple analysis methods. These results indicate that unlike some other viruses, for which an effect on the microbial composition was noted, SARS-CoV-2 does not have a strong effect on the nasopharynx microbial habitants.

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Larval Diet Abundance Influences Size and Composition of the Midgut Microbiota of Aedes aegypti Mosquitoes

Frontiers in Microbiology ◽

10.3389/fmicb.2021.645362 ◽

2021 ◽

Vol 12 ◽

Author(s):

Hannah J. MacLeod ◽

George Dimopoulos ◽

Sarah M. Short

Keyword(s):

Aedes Aegypti ◽

Bacterial Load ◽

Amplicon Sequencing ◽

Larval Diet ◽

Microbiota Composition ◽

Vector Species ◽

Larval Food ◽

Disease Vector ◽

16S Amplicon Sequencing ◽

The Impact

The midgut microbiota of the yellow fever mosquito Aedes aegypti impacts pathogen susceptibility and transmission by this important vector species. However, factors influencing the composition and size of the microbiome in mosquitoes are poorly understood. We investigated the impact of larval diet abundance during development on the composition and size of the larval and adult microbiota by rearing Aedes aegypti under four larval food regimens, ranging from nutrient deprivation to nutrient excess. We assessed the persistent impacts of larval diet availability on the microbiota of the larval breeding water, larval mosquitoes, and adult mosquitoes under sugar and blood fed conditions using qPCR and high-throughput 16S amplicon sequencing to determine bacterial load and microbiota composition. Bacterial loads in breeding water increased with increasing larval diet. Larvae reared with the lowest diet abundance had significantly fewer bacteria than larvae from two higher diet treatments, but not from the highest diet abundance. Adults from the lowest diet abundance treatment had significantly fewer bacteria in their midguts compared to all higher diet abundance treatments. Larval diet amount also had a significant impact on microbiota composition, primarily within larval breeding water and larvae. Increasing diet correlated with increased relative levels of Enterobacteriaceae and Flavobacteriaceae and decreased relative levels of Sphingomonadaceae. Multiple individual OTUs were significantly impacted by diet including one mapping to the genus Cedecea, which increased with higher diet amounts. This was consistent across all sample types, including sugar fed and blood fed adults. Taken together, these data suggest that availability of diet during development can cause lasting shifts in the size and composition of the microbiota in the disease vector Aedes aegypti.

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Phyllosphere community assembly and response to drought stress on common tropical and temperate forage grasses

10.1101/2021.05.10.443535 ◽

2021 ◽

Author(s):

Emily K. Bechtold ◽

Stephanie Ryan ◽

Sarah E. Moughan ◽

Ravi Ranjan ◽

Klaus Nüsslein

Keyword(s):

Microbial Community ◽

Drought Stress ◽

Bacterial Community ◽

Community Assembly ◽

Community Diversity ◽

Grass Species ◽

Severe Drought ◽

Hormone Production ◽

Plant Host ◽

Bacterial Community Diversity

Grasslands represent a critical ecosystem important for global food production, soil carbon storage, and water regulation. Current intensification and expansion practices add to the degradation of grasslands and dramatically increase greenhouse gas emissions and pollution. Thus, new ways to sustain and improve their productivity are needed. Research efforts focus on the plant-leaf microbiome, or phyllosphere, because its microbial members impact ecosystem function by influencing pathogen resistance, plant hormone production, and nutrient availability through processes including nitrogen fixation. However, little is known about grassland phyllospheres and their response to environmental stress. In this study, globally dominant temperate and tropical forage grass species were grown in a greenhouse under current climate conditions and drought conditions that mimic future climate predictions to understand if (i) plant host taxa influence microbial community assembly, (ii) microbial communities respond to drought stress, and (iii) phyllosphere community changes correlate to changes in plant host traits and stress-response strategies. Community analysis using high resolution sequencing revealed Gammaproteobacteria as the dominant bacterial class, which increased under severe drought stress on both temperate and tropical grasses while overall bacterial community diversity declined. Bacterial community diversity, structure, and response to drought were significantly different between grass species. This community dependence on plant host species correlated with differences in grass species traits, which became more defined under drought stress conditions, suggesting symbiotic evolutionary relationships between plant hosts and their associated microbial community. Further understanding these strategies and the functions microbes provide to plants will help us utilize microbes to promote agricultural and ecosystem productivity in the future.

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Uncovering the Structure and Function of Microbial Communities Formed During Periodic Tilling of TNT and DNT Co-Contaminated Soils

10.1101/2020.12.12.420737 ◽

2020 ◽

Author(s):

Saeed Keshani Langroodi ◽

Yemin Lan ◽

Ben Stenuit ◽

Gail Rosen ◽

Joseph B Hughes ◽

...

Keyword(s):

Microbial Community ◽

Bacterial Community ◽

Contaminated Soils ◽

Bacterial Community Composition ◽

Amplicon Sequencing ◽

Metagenomic Data ◽

Military Conflict ◽

Nitro Explosives ◽

And Function ◽

Aerobic And Anaerobic

Environmental contamination by 2,4,6-trinitrotoluene (TNT), historically the most widely used secondary explosive, is a long-standing problem in former military conflict areas and at manufacturing and decommissioning plants. In field test plots at a former explosives manufacturing site, removal of TNT and dinitrotoluenes (DNTs) was observed following periods of tillage. Since tilling of soils has previously been shown to alter the microbial community, this study was aimed at understanding how the microbial community is altered in soils with historical contamination of nitro explosives from the former Barksdale TNT plant. Samples of untilled pristine soils, untilled TNT-contaminated soils and tilled TNT-contaminated soils were subjected to targeted amplicon sequencing of 16S ribosomal RNA genes in order to compare the structure of their bacterial communities. In addition, metagenomic data generated from the TNT tilled soil was used to understand the potential functions of the bacterial community relevant to nitroaromatic degradation. While the biodiversity dropped and the Burkholderiales order became dominant in both tilled and untilled soil regardless of tillage, the bacterial community composition at finer taxonomic levels revealed a greater difference between the two treatments. Functional analysis of metagenome assembled genome (MAG) bins through systematic review of commonly proposed DNT and TNT biotransformation pathways suggested that both aerobic and anaerobic degradation pathways were present. A proposed pathway that considers both aerobic and anaerobic steps in the degradation of TNT in the scenario of the tilled contaminated soils is presented.

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PSI-10 Establishing a foundation to harvest the potential of the microbiome in poultry production

Journal of Animal Science ◽

10.1093/jas/skz258.594 ◽

2019 ◽

Vol 97 (Supplement_3) ◽

pp. 293-294

Author(s):

Camila S Marcolla ◽

Benjamin Willing

Keyword(s):

Microbial Community ◽

Gut Microbiota ◽

Community Composition ◽

Relative Abundance ◽

Microbial Community Composition ◽

Alpha Diversity ◽

Poultry Production ◽

Microbiota Composition ◽

Microbial Composition ◽

The Impact

Abstract This study aimed to characterize poultry microbiota composition in commercial farms using 16S rRNA sequencing. Animals raised in sanitized environments have lower survival rates when facing pathogenic challenges compared to animals naturally exposed to commensal organisms. We hypothesized that intensive rearing practices inadvertently impair chicken exposure to microbes and the establishment of a balanced gut microbiota. We compared gut microbiota composition of broilers (n = 78) and layers (n = 20) from different systems, including commercial intensive farms with and without in-feed antibiotics, organic free-range farms, backyard-raised chickens and chickens in an experimental farm. Microbial community composition of conventionally raised broilers was significantly different from antibiotic-free broilers (P = 0.012), from broilers raised outdoors (P = 0.048) and in an experimental farm (P = 0.006) (Fig1). Significant community composition differences were observed between antibiotic-fed and antibiotic-free chickens (Fig2). Antibiotic-free chickens presented higher alpha-diversity, higher relative abundance of Deferribacteres, Fusobacteria, Bacteroidetes and Actinobacteria, and lower relative abundance of Firmicutes, Clostridiales and Enterobacteriales than antibiotic-fed chickens (P < 0.001) (Fig3). Microbial community composition significantly changed as birds aged. In experimental farm, microbial community composition was significant different for 7, 21 and 35 day old broilers (P < 0.001), and alpha diversity increased from 7 to 21d (P < 0.024), but not from 21 to 35d; whereas, in organic systems, increases in alpha-diversity were observed from 7d to 21d, and from 21d to 35d (P < 0.05). Broilers and layers raised together showed no differences in microbiota composition and alpha diversity (P > 0.8). It is concluded that production practices consistently impact microbial composition, and that antibiotics significantly reduces microbial diversity. We are now exploring the impact of differential colonization in a controlled setting, to determine the impact of the microbes associated with extensively raised chickens. This study will support future research and the development of methods to isolate and introduce beneficial microbes to commercial systems.

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Hill-based Dissimilarity Indices and Null Models for Analysis of Microbial Community Assembly

10.21203/rs.3.rs-33130/v1 ◽

2020 ◽

Author(s):

Oskar Modin ◽

Raquel Liebana ◽

Soroush Saheb-Alam ◽

Britt-Marie Wilén ◽

Carolina Suarez ◽

...

Keyword(s):

Microbial Community ◽

Microbial Communities ◽

Community Assembly ◽

Null Model ◽

Amplicon Sequencing ◽

Null Models ◽

Marker Genes ◽

Experimental Systems ◽

Microbial Community Assembly ◽

The Impact

Abstract Background: High-throughput amplicon sequencing of marker genes, such as the 16S rRNA gene in Bacteria and Archaea, provides a wealth of information about the composition of microbial communities. To quantify differences between samples and draw conclusions about factors affecting community assembly, dissimilarity indices are typically used. However, results are subject to several biases and data interpretation can be challenging. The Jaccard and Bray-Curtis indices, which are often used to quantify taxonomic dissimilarity, are not necessarily the most logical choices. Instead, we argue that Hill-based indices, which make it possible to systematically investigate the impact of relative abundance on dissimilarity, should be used for robust analysis of data. In combination with a null model, mechanisms of microbial community assembly can be analyzed. Here, we also introduce a new software, qdiv, which enables rapid calculations of Hill-based dissimilarity indices in combination with null models.Results: Using amplicon sequencing data from two experimental systems, aerobic granular sludge (AGS) reactors and microbial fuel cells (MFC), we show that the choice of dissimilarity index can have considerable impact on results and conclusions. High dissimilarity between replicates because of random sampling effects make incidence-based indices less suited for identifying differences between groups of samples. Determining a consensus table based on count tables generated with different bioinformatic pipelines reduced the number of low-abundant, potentially spurious amplicon sequence variants (ASVs) in the data sets, which led to lower dissimilarity between replicates. Analysis with a combination of Hill-based indices and a null model allowed us to show that different ecological mechanisms acted on different fractions of the microbial communities in the experimental systems.Conclusions: Hill-based indices provide a rational framework for analysis of dissimilarity between microbial community samples. In combination with a null model, the effects of deterministic and stochastic community assembly factors on taxa of different relative abundances can be systematically investigated. Calculations of Hill-based dissimilarity indices in combination with a null model can be done in qdiv, which is freely available as a Python package (https://github.com/omvatten/qdiv). In qdiv, a consensus table can also be determined from several count tables generated with different bioinformatic pipelines.

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To rarefy or not to rarefy: Enhancing microbial community analysis through next-generation sequencing

10.1101/2020.09.09.290049 ◽

2020 ◽

Author(s):

Ellen S. Cameron ◽

Philip J. Schmidt ◽

Benjamin J.-M. Tremblay ◽

Monica B. Emelko ◽

Kirsten M. Müller

Keyword(s):

Microbial Community ◽

Community Analysis ◽

Amplicon Sequencing ◽

Microbial Community Analysis ◽

List Type ◽

Sequencing Data ◽

Water And Wastewater Treatment ◽

Library Size ◽

Size Selection ◽

The Impact

AbstractThe application of amplicon sequencing in water research provides a rapid and sensitive technique for microbial community analysis in a variety of environments ranging from freshwater lakes to water and wastewater treatment plants. It has revolutionized our ability to study DNA collected from environmental samples by eliminating the challenges associated with lab cultivation and taxonomic identification. DNA sequencing data consist of discrete counts of sequence reads, the total number of which is the library size. Samples may have different library sizes and thus, a normalization technique is required to meaningfully compare them. The process of randomly subsampling sequences to a selected normalized library size from the sample library—rarefying—is one such normalization technique. However, rarefying has been criticized as a normalization technique because data can be omitted through the exclusion of either excess sequences or entire samples, depending on the rarefied library size selected. Although it has been suggested that rarefying should be avoided altogether, we propose that repeatedly rarefying enables (i) characterization of the variation introduced to diversity analyses by this random subsampling and (ii) selection of smaller library sizes where necessary to incorporate all samples in the analysis. Rarefying may be a statistically valid normalization technique, but researchers should evaluate their data to make appropriate decisions regarding library size selection and subsampling type. The impact of normalized library size selection and rarefying with or without replacement in diversity analyses were evaluated herein.Highlights▪ Amplicon sequencing technology for environmental water samples is reviewed▪ Sequencing data must be normalized to allow comparison in diversity analyses▪ Rarefying normalizes library sizes by subsampling from observed sequences▪ Criticisms of data loss through rarefying can be resolved by rarefying repeatedly▪ Rarefying repeatedly characterizes errors introduced by subsampling sequences

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